Method to identify a surface-bound molecule

ABSTRACT

The present invention relates to a method of identifying a molecule of a molecule-substrate complex, wherein the molecule is covalently attached directly to a substrate or indirectly by means of a linking moiety, comprising: (a) bombarding the molecule-substrate complex with energized particles to cleave the molecule from the molecule-substrate complex; and (b) determining the molecular weight of the cleaved molecule by means of mass spectrometry. The inventive method may further comprise irradiating the cleaved molecule with photons.

[0001] The present invention claims benefit of U.S. ProvisionalApplication No. 60/004,702, filed Oct. 3, 1995.

[0002] This invention was made with U.S. Government support underContract No. CHE-9115011. The U.S. Government has certain rights in thisinvention.

[0003] The present invention relates to the field of biophysicalanalysis of molecules. In particular, the present invention is usefulfor the identification and analysis of a molecule, such as a member of acombinatorial library, wherein the identified molecule has ademonstrated pharmacological or physiological activity.

[0004] Over the past ten years, there has been a growing demand for theproduction and identification of molecules that have pharmacological orother physiological activity as, for example, agonists or antagonists ofvarious cellular acceptor molecules, such as cell-surface receptors,enzymes, or antibodies. Such molecules can be peptides,oligonucleotides, or other organic compounds, such as heterocyclics andthe like, which are commonly the products of combinatorial synthesis,thus forming the members of combinatorial libraries. The unifyingfeature of these molecules is operational in that they bind specificallyto known acceptors. In consequence of such binding, a physiologicalresponse occurs whereby certain biological processes are modulated,which can have applications in medicine and agriculture.

[0005] Searching for molecules that are useful in medical or veterinaryapplications, or in agriculture or agrobiology, entails (1) generatingcollections of such molecules, (2) screening such molecules forphysiological activity, and (3) identifying the structure of moleculesthat provide a positive result in the screen. The first two steps can beaccomplished using methods well-known in the art, some of which werediscussed in Benkovic et al., PCT/US95/03355, which is incorporatedherein in toto by reference. The subject matter of Benkovic et al.related to a mass spectrometric method for identification of suchmolecules, including those that are members of a combinatorial library.Covalent attachment of such molecules to a substrate, such as apolystyrene or other resin particle, via a suitable linker, thus forminga molecule-substrate complex, is commonly used for purposes ofmanipulating the molecules; however, for mass spectrometricidentification of the molecules, the molecules must be separated fromthe substrate. One approach that has been used requires use of a photo-or acid-labile linker, however such an approach requires identificationof linkers that include either a photo-labile or an acid-labile linkageand subjecting the substrate-attached molecules to a suitable wavelengthof light or acid. Such a step requires extra manipulation of thesamples, and consumes time. Secondly, the process used to cleave thecovalent linkage between the molecule and the molecule-substrate complexmay destroy or damage the substrate or the entire molecule-substratecomplex, thus retesting of a given molecule requires having additionalmolecule-substrate complexes.

[0006] Accordingly, the analysis of a molecule covalently linked to asubstrate, such as the molecules of a combinatorial library, or anycollection of molecules so linked to a substrate or substrates, isnecessarily impeded by the rate at which substrates, such as beads,having individual molecules attached thereto can be analyzed for theidentity of the attached molecule. In view of the literally millions ofcandidate molecules to be screened in a given library, for example, itis probable that at least hundreds, if not thousands, of themolecule-attached beads would generate positive signals (including falsepositive signals) requiring further analysis. The limitation of beingable to sequence only a few molecules per day, as has been reported byLam et al., Nature, 354, 82 (1991), for example, therefore, presents astrong drawback to current strategies of screening collections ofmolecules, such as combinatorial libraries, for pharmaceuticalcompounds. Moreover, if a method allowed identification of a moleculeincluded on a molecule-substrate complex with respect to molecularweight, more preferably with respect to structure, where the moleculewas indicated in a screen as having a desireable characteristic, withouthaving to remove such a molecule-substrate complex from the group ofother such complexes, in the presence of which the molecule-substratecomplex was screened, the procedure of screening and identifyingmolecules of interest would be greatly improved. Further yet, if amethod required essentially a unified step of testing amolecule-substrate complex directly without causing damage to thecomplex but for the removal of a portion of the included molecule, theprocess would not only be faster, but would preserve themolecule-substrate complex having a then reduced amount of the molecule,which could be used for further analysis.

SUMMARY OF THE INVENTION

[0007] It has now been discovered that a mass spectrometric assay can beused to analyze molecules covalently attached to a substrate, suchmolecules being peptides, oligonucleotides, heterocyclic molecules, orother chemical species. Such molecules can be members of any collectionof molecules including those isolated from natural sources orsynthesized, such as those of a combinatorial library. Individualmembers of a collection of molecules, for example, can be constructed onor attached to a suitable substrate or substrates and screened, and theindividual substrate or portion thereof that is identified as having amolecule that, for example, specifically interacts with an acceptormolecule of interest (i.e., positive screen result) can be identified inthe presence of identical substrates having other unselected moleculesattached thereto and subjected to mass spectrometric assay withoutremoval from the total collection to determine the precise molecularweight of the selected molecule. A preferred aspect of the methodincludes the use of linking moieties or substrates having reactivegroups attached thereto that covalently link the individual molecules ofthe collection to the substrate, whereby the linkage of at least aportion of the molecules linked to the substrate is cleaved withoutdisturbing the molecule's structure or the integrity of the substrate,allowing analysis of the free molecules and subsequent analysis of theremaining linked molecules. Consequently, the present invention greatlyimproves the ability of artisans of the relevant art to identify, forexample, pharmaceutically active agents derived from collections ofmolecules, such as combinatorial libraries.

[0008] In particular, the present invention relates to a method ofidentifying a molecule of a molecule-substrate complex, wherein themolecule is covalently attached directly to a substrate or indirectly bymeans of a linking moiety, comprising:

[0009] (a) bombarding the molecule-substrate complex with energizedparticles to cleave the molecule from the molecule-substrate complex;and

[0010] (b) determining the molecular weight of the cleaved molecule bymeans of mass spectrometry. Preferably, the method further comprisesirradiating the cleaved molecule with photons.

[0011] The present method is further directed to a molecule that isselected from the group consisting of amino acids, peptides,oligonucleotides, heterocyclic compounds, and combinations thereof. Thesubstrate used in the context of the present invention preferablycomprises a polymeric resin or a metal; and, in another embodiment,further comprises a linking moiety attached thereto. Preferably, thepolymeric resin is a polystyrene resin having a linking moiety attachedthereto.

[0012] The linking moiety used in the context of the present inventionpreferably comprises at least one reactive group that is selected fromthe group consisting of hydroxyl, amino, carboxyl, acetal, thioacetal,C₁-C₁₀ alkylamino, C₁-C₁₀ aralkylamino, and C₁-C₁₀ haloalkyl, and ano-nitrobenzylic group having a benzylic hydrogen. Preferably, thelinking moiety is selected from the group consisting of F-moc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine,F-moc-methoxy-4′(gamma-carboxypropyloxy) benzhydrylamine, p-alkoxybenzylalcohol, benzylacetal, benzylthioacetal, benzhydrylamine, Cl—CH₂—Ph,2-methoxy-4-alkoxy benzyl alcohol, and o-nitrobenzyloxy carbonyl. Morepreferably, the linking moiety is selected from the group consisting of2-methoxy-4-alkoxy benzyl alcohol, benzylacetal, and benzylthioacetal.

[0013] The present method includes cleaving the molecule from thesubstrate in the molecule-substrate complex without substantialmodification of the molecule or destruction of the substrate. Suchcleaving is preferably accomplished by bombardment of themolecule-substrate complex with energized particles, wherein theparticles are preferably gallium or argon. The particles are energizedby subjection to an electric field of between about one and about 30kilovolts. It is believed that the free molecule that results from thebombardment is charged or uncharged; the molecule becomes charged orfurther charged by irradiation by a laser beam.

[0014] The substrate used in the context of the present method ispreferably a bead. A bead used in this context has a diameter of fromabout 10 microns to about 120 microns.

[0015] The mass spectrometry used in the present invention is preferablytime-of-flight secondary ion mass spectrometry. The method furthercomprises mapping of the spatial distribution of the molecules on theaforementioned beads that, for example, are arranged on a grid.

[0016] In a preferred embodiment, the molecule subjected to analysisunder the present invention is an amino acid or a peptide. Preferably,the peptide comprises two to ten amino acids. The method furthercomprises determination of the sequence of the peptide from thefragmentation pattern obtained in the mass spectrometry. Alternatively,the molecule subjected to analysis under the present invention is aheterocyclic compound comprising four to seven membered rings having N,S, or O, and combinations thereof.

[0017] A preferred embodiment of the present invention relates to amethod of identifying a molecule of a molecule-substrate complex,wherein the molecule is covalently attached directly to a substrate orindirectly by means of a linking moiety, comprising:

[0018] (a) bombarding the molecule-substrate complex with energizedparticles to cleave the molecule from the molecule-substrate complex;

[0019] (b) irradiating the cleaved molecule with photons; and

[0020] (c) determining the molecular weight of the irradiated moleculeby means of mass spectrometry, wherein the substrate is a polystyrenebead having a reactive group, the molecule is an amino acid, peptide,oligonucleotide, or a heterocyclic compound, or a combination thereof,the covalent bond is sensitive to energized particle bombardment, theenergized particles are gallium atoms, the photon source is a laser, andthe mass spectrometry is time-of-flight secondary ion mass spectrometry.

[0021] These and other features and advantages of the invention will bemore readily apparent upon reading the following detailed description ofthe invention and upon reference to the accompanying drawings, all ofwhich are given by way of illustration only, and are not limitative ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 depicts various linking moieties attaching angiotensin IIreceptor antagonist to various polystyrene substrates.

[0023]FIG. 2 is a graph depicting time-of-flight distributions ofintensity (y-axis) over time-of-flight (μs; x-axis) ofphenethylmercaptan subsequent to impact of H₂ ⁺ and Ar⁺.

[0024]FIG. 3 is a graph depicting kinetic energy distributions ofintensity (y-axis) over kinetic energy (eV; x-axis) ofphenethylmercaptan at 300 K and 184 K, shown with correspondingMaxwell-Boltzmann fit.

[0025]FIG. 4 is a graph depicting time-of-flight distributions ofintensity (y-axis) over time-of-flight (μs; x-axis) of golddimer shownwith the high energy component of phenethylmercaptan.

DETAILED DESCRIPTION

[0026] The following detailed description of the instant invention isprovided to aid those skilled in the art in practicing the presentinvention, but should not be construed to limit the present invention,as modifications and variations in the embodiments herein discussed maybe made by those of ordinary skill in the art without departing from thespirit or scope of the present inventive discovery.

[0027] The following terms shall have the definitions provided herewith:

[0028] collection—a set of molecules, which may be present asmolecule-substrate complexes, such as a combinatorial library, but notlimited thereto.

[0029] linker—a linking moiety used for indirect attachment of amolecule to a substrate, or the functional group used for directattachment of a molecule to a substrate.

[0030] linking moiety—a chemical unit that is covalently attached to themolecule and attached to the substrate.

[0031] linking substrate—a substrate comprising a reactive group towhich a molecule can attach covalently thereto.

[0032] molecule—a chemical unit composed of one or more atoms, which mayor may not be charged.

[0033] molecule-substrate complex—a substrate having a moleculecovalently attached thereto, either directly via a functional group onthe substrate or indirectly via a linking moiety.

[0034] substrate—a surface to which a molecule can be attached, eitherdirectly by a reactive group included with the substrate, as in alinking substrate, or indirectly by a linking moiety.

[0035] The present invention provides a method used in the inventivemethod that greatly improves the ability of an ordinary artisan toidentify and characterize a molecule that is attached to a surface.Molecules of particular interest that can be identified in the contextof the present invention include amino acids, peptides,oligonucleotides, heterocyclic compounds, combinations thereof, and thelike. Such molecules include, in particular, pharmaceutically-activemolecules, which can be members of collections of synthesized moleculesor isolated natural molecules. Synthetic molecules include thoseprepared by combinatorial chemical methods known to the art, whichcollections are commonly referred to as combinatorial libraries. Themembers of such a collection can be constructed in association with orattached to a suitable substrate, such as a polystyrene bead surface.Such association between the molecules and the substrates, referred toherein as molecule-substrate complexes, are preferably mediated bycovalent linkage, particularly during the construction of combinatoriallibraries, for example. Preferably, the covalent linkage is cleavedusing means that does not modify or substantially modify the structureof the linked molecule, nor does the means modify or substantiallymodify the substrate. A molecule or substrate that is not substantiallymodified by the cleavage of the molecule from the molecule-substratecomplex has, with respect to the molecule, a molecular weight asmeasured by mass spectrometry that provides sufficient information suchthat the molecular weight of the molecule on the substrate can bededuced, i.e., positive results, and with respect to the substrate,retains a sufficient quantity of attached molecules such that asubsequent mass spectrometric run directed at the molecule-substratecomplex would provide positive results.

[0036] Molecules from any of the aforementioned collections preferablyare provided as molecule-substrate complexes, as discussed herein. Thelinker used to form the molecule-substrate complexes must be selectedfor linkers that provide a covalent linkage to the molecule that willpreferentially be cleaved by the ion beam of the mass spectrometricmethod of the present invention. By preferential cleavage of the linker,it is intended that at least about 5% of the cleavage events caused bythe ion beam occur at a covalent bond or bonds within the linker orbetween the linker and the molecule. Prefered linkers are associatedwith preferential cleavage of at least about 20%. More prefered linkersare associated with preferential cleavage of at least about 50%.

[0037] The molecules of a preferred collection are linked covalently tothe substrate, using methods well known in the art, thus forming amolecule-substrate complex. A preferred covalent linkage between themolecule and the substrate has the characteristic of being able to breakin response to external changes caused by energized particles at levelsthat do not modify or do not substantially modify the structure of themolecules or the substrates of the collection. Such a covalent linkagemay be effected, for example, by means of a suitable linking moiety thatcouples both to the molecule and the substrate. The substrate itself caninclude suitable reactive groups coupled thereto, such that such alinking substrate links to a molecule without need of a separate linkingmoiety. Examples of such linking substrates are included in FIG. 1. Whena linking moiety or a linking substrate is used, the covalent bondsbetween the molecule and the substrate will break in consequence of theenergized particles at one or more of the covalent bonds associated withthe linking moiety or the aforementioned reactive groups of the linkingsubstrate, thereby destroying any covalent linkage between the moleculeand the substrate. At least an appreciable proportion of the populationof molecules will be fully free of the covalent linkage, however, someor even a majority of the molecules may remain attached covalently.

[0038] As noted above, the covalent linkage is broken by bombarding themolecule-substrate complex with energized particles. Suitable particlesinclude, without limitation, gallium or argon atoms, which are energizedby subjection of such or other particles to an electric field betweenabout one and about 30 kilovolts. Such molecules so freed of theircovalent linkage to the substrate typically are uncharged. However, inessentially the same step, an uncharged free molecule is ionized byirradiation by a suitable ionization means, such as a laser beam, asdiscussed herein.

[0039] Suitable linking moieties or linking substrates are those thatcomprise a reactive functional group selected from the group consistingof alcohol, amino, carboxyl, acetal, thioacetal, and aminoalkyl,aralkyl, amino aralkyl, and haloalkyl, and a nitroaromatic group havinga benzylic hydrogen ortho to the nitro group, such as o-nitrobenzylderivatives, and benzylsulfonyl derivatives; and which covalent bondformed with such a group is cleavable by exposure to a suitableenergized particle, as discussed above. Preferably, the linking moietyor linking substrate comprises at least one reactive group that isselected from the group consisting of hydroxyl, amino, carboxyl, acetal,thioacetal, C₁-C₁₀ alkylamino, C₁-C₁₀ aralkylamino, and C₁-C₁₀,haloalkyl, and an ortho-nitrobenzylic group having a benzylic hydrogen.

[0040] In particular, suitable linking moieties include p-alkoxybenzylalcohol (used in the Wang resin),F-moc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine,F-moc-4-methoxy-4′-(gamma-carboxypropyloxy)-benzhydrylamine,4-hydroxymethyl-phenoxy-acetic acid, aminomethyl (used in the PAMresin), benzhydrylamine, Cl—CH₂—Ph—(used in Merrifield resin),benzylacetal (used in the Acetal resin), benzylthioacetal (used in theThioacetal resin), and 2-methoxy-4-alkoxybenzyl alcohol (used in Sasrin®resin). See FIG. 1. Preferred linking moieties include F-moc2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine,F-moc-methoxy-4′(gamma-carboxypropyloxy) benzhydrylamine, p-alkoxybenzylalcohol, benzhydrylamine, Cl—CH₂Ph, 2-methoxy-4-alkoxy benzyl alcohol,6-nitroveratryloxy carbonyl, 2-nitrobenzyloxycarbonyl, and α,α-dimethyl-dimethoxybenzyloxycarbonyl, more preferred linking moieties include2-methoxy-4-alkoxybenzyl alcohol. It is appreciated that differentlinker chemistry may enhance the molecular ion signal of covalentlyattached species.

[0041] The covalent linkage between the substrate and the molecule mayalso be mediated by a reactive group or groups attached to thesubstrate, as in the aforementioned linking substrate. For example, asrecited above, the polystyrene-derivative bead known as Sasrin® (BachemBiosciences) has a reactive group (2-methoxy-4-alkoxy benzyl alcohol)that covalently couples to carboxylic acid groups found on all peptides.See FIG. 1.

[0042] Construction of a combinatorial library or any of the othercollections herein noted is known to the art and described elsewhere,such as Benkovic et al., PCT/US95/03355 and Lam et al. (supra), asexamples. Screening of such collections is also described in theaforementioned references. A preferred identification approach wouldtake into account the fact that molecule-substrate complexes thatinclude, for example, peptides, oligonucleotides, or heterocycliccompounds can be constructed such that the molecules are desorbed intactor substantially intact from a substrate, particularly from a beadsurface, even when covalently attached to the substrate initially.Because each bead, for example, may have adsorbed thereto only afemtomole quantity of a particular molecule, or less, and only a portionof the particular molecule is desorbed in a given analytical run,extreme sensitivity of the method of analysis is required. For example,a standard 40 micron sphere covered with one layer of phenylalanine willonly have about 50 femtomoles of surface molecules available forsampling. With sufficient sensitivity of the analytical procedure, andthe capability of preserving a given molecule-substrate complex after aportion of the included molecules have been removed, provides a valuablemethod for analyzing and re-analyzing particular molecules.

[0043] The present method measures the molecular weight of suchmolecules upon their removal from the substrate and subsequentionization. Removal from the substrate is accomplished by application ofa suitable ionic beam, such as one resulting by subjecting gallium orargon, for example, to an electric field of between about one and about30 kilovolts. If the so removed molecule is uncharged, it becomescharged by passing through a suitable laser beam, as is known in theart. A suitable laser has a pulse length of about 10 nanoseconds orless, a wavelength in the ultraviolet range, and produces energy perpulse of about 1 to about 10 millijoules. Alternatively, if the soremoved molecule is charged, the laser is unnecessary, although it mayserve to increase the charge on the molecule. The remaining step of themethod employs any suitable design of mass spectrometry for determiningmolecular weight of the ionized molecule.

[0044] The method preferably employs imaging secondary ion massspectrometry to identify the molecular weights of molecules adsorbed tothe polystyrene bead surfaces, such as magnetic sector SIMS, quadrupoleSIMS, Fourier Transformation SIMS, or time-of-flight SIMS (TOF-SIMS).The methodology actually used for any given SIMS analysis is known inthe art, and may vary both with the machine used and artisan operatingthe machine. Preferably, the present invention employs TOF-SIMS.Detection of the mass of (secondary ions formed in a TOF-SIMS protocolallows the unique identification of the corresponding library member,presuming that the method of construction of the library is known sothat an artisan can assign discrete molecular weights to all moleculesso generated and ionization fragments thereof (generated in the TOF-SIMSmethod).

[0045] In TOF-SIMS, a pulsed beam of primary ions is directed to asample surface. The arriving primary ions desorb molecules of the samplepresent in a monolayer at the surface of the sample. Molecules that wereattached to the surface by a covalent linkage, for example, and are sodesorbed are typically uncharged in that no mass spectrometric profileresults in the absence of an ionization means. Such molecules becomecharged by a suitable ionization means, such as a laser, which ispositioned in an instrument by which the present method can be performedsuch that any such uncharged desorbed molecules become charged. Forexample, uncharged desorbed molecules, upon receiving thermal energy,can evaporate and immediately migrate into a laser beam, by which theybecome charged, thus forming secondary ions. These generated secondaryions are then accelerated to a uniform energy by an electric field, anddrift through a fixed distance to a detector. The time-of-flight ofthese uniform energy particles through the fixed distance is directlyproportional to the charge-to-mass ratio (m/z) of the ion. Because onlythe time-of-flight of an ion is measured to determine its mass, TOF-SIMSprovides for parallel detection of all masses present in a sample, andan effectively unlimited mass detection range with high mass resolution.Indeed, TOF-SIMS, provides a 10⁴-10₆ fold improvement in sensitivityover scanning mass spectrometric methods employing other detectors, suchas magnetic sector fields and quadrapoles, which are well known in theart. TOF-SIMS thus provides a direct mass spectrometric assay that isgenerally applicable to reading a wide variety of molecules assembled ina collection, such as a combinatorial library.

[0046] The considerations relevant to use of TOF-SIMS for such assaysare discussed in the literature. For example, as discussed by Winogradin Ion Beams and Laser Postionization for Molecule-Specific Imaging(Anal. Chem., 65, 622A-629A (1993)), an energetic primary ion bombardinga sample on a solid surface creates a large amount of damage within 50Angstroms of the point of impact. Unless the dose of incident ions iskept below approximately 1% of the number of sample molecules forming amonolayer, the ion bombardment alters the surface chemistry. The dose ofincident ions of 1% is referred to as the “static limit.” In TOF-SIMS,the dosage of primary ions remains below the static limit because theincident ion beam is directed toward the sample as a very short pulse.Use of a pulsed incident beam is also advantageous because a spectrumwith a dynamic range of several orders of magnitude can be obtained bythe accumulation of a large number of cycles with high repetition rates,as discussed by Benninghoven et al. in Surface MS: Probing Real-WorldSamples (Anal. Chem., 65, 630A-639A (1993)). Increased sensitivity mayalso be realized using special cationization schemes or by laserpostionization of sputtered neutral molecules, as discussed by Winogradet al., Inst. Phys. Conf. Ser., 128, 259 (1992).

[0047] The TOF-SIMS technique also allows the primary ion beam to befocused to a spot size of less than 150 nm, thereby allowing theconcentration of molecules to be mapped over spatial domains byrastering or moving the ion beam across pixels defined on the sample andtaking spectra at each pixel. Other aspects of TOF-SIMS imaging arediscussed by Chait and Standing in Time-of-Flight Mass Spectrometer forMeasurement of Secondary Ion Mass Spectra (Int. J. Mass Spectrom. IonPhys., 40, 185-193 (1981)); and by Steffens et al. in A Time-of-FlightMass Spectrometer for Static SIMS Applications (J. Vac. Sci. Technol., A3(3), 1322 (1985)).

[0048] In certain situations, the information obtained by TOF-SIMS maynot fully distinguish and identify all members of a combinatoriallibrary. For example, various isomers of a given peptide may be present,each having the same mass, as, for example, in the case ofphenylalanine-glycine-leucine and glycine-leucine-phenylalanine. In suchsituations, TOF-SIMS can be used to determine the sequence of theselected peptide nonetheless, provided that the library was constructedfrom a known set of building blocks. As discussed by Poppe-Schriemer etal. in Sequencing an “Unknown” Peptide by Time-of-Flight Secondary IonMass Spectrometry (Int. J. Mass Spectrom. Ion Phys., 111, 301-315(1991)), the parent ions subjected to TOF-SIMS necessarily break down tothe various fragment ions, the masses of which can be compared andanalyzed based on existing mass data to determine the structure of theselected peptide. This procedure is effective to the extent that theselected molecule is one of the possible peptides of the combinatoriallibrary as determined by the construction of the library. This procedureis also limited by the resolving power of TOF-SIMS to distinguish suchfragmentions (TOF-SIMS mass accuracy is currently on the order of ±0.01amu, according to Winograd, supra).

[0049] Alternatively, an isotope indexing scheme can be used todifferentiate between molecules that otherwise have the same mass. Forexample, to differentiate between phenylalanine-glycine-leucine andglycine-leucine-phenylalanine, one can either examine the fragmentationpattern in the SIMS spectrum or synthesize one of the peptides usingleucine having ¹⁵N, an isotope that is readily distinguished in TOF-SIMSas its atomic mass is increased by one unit. Distinguishing between aleucine and an isoleucine residue, which are isomers, necessarily wouldrequire such an alternate method. Similarly, one could usedifferentially L and D amino acids, using methods well known in the art.

[0050] In particular, the present invention relates to a method ofidentifying a molecule of a molecule-substrate complex, wherein themolecule is covalently attached directly to a substrate or indirectly bymeans of a linking moiety, comprising: (a) bombarding themolecule-substrate complex with energized particles to cleave themolecule from the molecule-substrate complex; and (b) determining themolecular weight of the cleaved molecule by means of mass spectrometry.Preferably, the mass spectrometry that is utilized in the context of thepresent invention is TOF-SIMS, as noted above. The molecules can be anysuitable molecules, such as, without limitation, at least one of thegroup consisting of amino acids, peptides, oligonucleotides, orheterocyclic compounds. Such molecules can be synthetic, such as thoseof a combinatorial library, or isolated from nature. In one embodiment,the present method is applicable to a collection of molecules comprisingamino acids that are naturally occurring or synthetic. A preferredcollection of molecules is a combinatorial library that has moleculesthat are peptides or heterocyclic compounds; a more preferred collectionof molecules is a combinatorial library that has molecules that arepeptides.

[0051] Suitable peptides comprise as few as two amino acids to as manyas about 50; preferably, suitable peptides comprise from about two aminoacids to about 20; most preferably, suitable peptides comprise fromabout two amino acids to about ten. Any amino acid may be incorporatedinto peptides screened and identified using the present invention,including any combination of the naturally occurring proteinogenic aminoacids as well as amino acids not naturally occurring in proteins suchas, but not limited to, dextrorotatory forms of the known amino acids,for example.

[0052] Suitable oligonucleotides consist of as few as two nucleotides toas many as about 50; preferably, suitable oligonucleotides consist offrom about five nucleotides to about 30; most preferably, suitableoligonucleotides consist of from about five oligonucleotides to about15. Any nucleotide may be incorporated into an oligonucleotide screenedand identified using the present invention, including any combination ofthe naturally occurring deoxyribonucleotides and ribonucleotides as wellas those not naturally occurring in biological systems, such as, but notlimited to, H-phosphonate derivatives,N-blocked-5′-O-DMT-deoxynucleoside3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,N-blocked-5′-O-DMT-deoxynucleoside3′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites,N-blocked-5′-O-DMT-deoxynucleoside 3′-(methyl-N,N-diisopropyl)phosphoramidites, N-blocked-5′-O-DMT-deoxynucleoside 3′-(2-chlorophenyl)phosphates, N-blocked-5′-O-DMT-deoxynucleoside 3′-(2-chlorophenyl2-cyanoethyl) phosphate, all of which are nucleoside derivatives used inoligonucleotide synthesis.

[0053] Suitable heterocyclic compounds consist of, at minimum, a singlethree membered ring to as much as a multiple of three membered orgreater membered rings coupled by carbon chains of 1 to about 20 atomsin length, such chains being saturated or not. Preferably, suitableheterocyclic compounds include a single three- to seven-membered ring,as well as, but not limited to varying combinations of three-, four-,five-, six-, or seven-membered rings having varying numbers of N, S, orO atoms. More preferably, suitable heterocyclic compounds includebenzodiazepine and derivatives thereof (as, for example, disclosed inBunin et al., J. Am. Chem. Soc., 114, 10997-10998 (1992)), penicillins,cephalosporins, and folate derivatives. most preferred, suitableheterocyclic compounds include benzodiazepine and derivatives thereof,and angiotensin II receptor antagonists. For example, one angiotensin IIreceptor antagonist that was developed to block the renin-angiotensinsystem for the treatment of heart failure and possibly chronic renalfailure (see, Weinstock et al., J. Med. Chem., 34, 1514 (1991); Keenanet al., J. Med. Chem., 36 1880 (1993)) can be identified in a mixture ofother heterocyclic compounds using the present invention. The formula ofthe aforementioned angiotensin II receptor antagonist, ethyl2-(2′-thiophenylmethyl)-3-[5′-{(1′-p-carboxyphenylmethyl)-2′-n-butyl}-imidazolyl]-propenoate,covalently linked to polystyrene beads through various linking moietiesis set forth in FIG. 1. The present invention may be applied to theidentification of derivatives of such compounds as benzodiazepine andthe noted angiotensin II receptor antagonist.

[0054] Mixed collections of molecules comprising amino acids, peptides,oligonucleotides, and heterocyclic compounds may be prepared byfollowing standard methods known to one of ordinary skill in the art,such as relates to combinatorial libraries, for example. Anoligonucleotide can be, for instance, linked to a peptide through the5′-hydroxyl of the oligonucleotide. The peptide end can be modified toinclude a carboxyl group. A process of esterification of the carboxylgroup with the 5′-hydroxyl of the oligonucleotide is used to produce amixed library containing peptide-oligonucleotide molecules. Brenner etal., (Proc. Nat'l Acad. Sci. USA, 89, 5381-5383 (1992) also describes amethod of preparation of mixed libraries having nucleotides andpeptides. A mixed library comprising a heterocyclic compound and apeptide is also prepared by the reaction of suitable functional groupspresent on the heterocyclic compound. For instance, the carboxyl groupon a heterocyclic compound is reacted with the amino group on thepeptide to provide an amide linkage.

[0055] The substrate upon or with which the molecules of thecombinatorial library are synthesized and/or associated may be anysuitable substrate, including, but not limited to, a suitable resin,such as polystyrene, Sasrin®, Wang resin, Pam resin, and Merrifieldresin, some of which are set forth in FIG. 1 and are known to the art,and a suitable metal, such as but not limited to gold, further includingsuitable combinations thereof. Suitable resins or metals are thoseresins or metals that can covalently attach to a molecule of theaforementioned collections, can be manipulated physically for thepurpose of moving the so attached molecules, and can withstand exposureto the ion beam used in the mass spectrometry used in the context of thepresent invention without becoming substantially damaged. Such resins ormetals are commercially available from Bachem Bioscience Inc., forexample. The substrate used in the present invention may be formed intoany suitable shape, including, but not limited to, spheres, cubes,rectangular prisms, pyramids, cones, ovoids, sheets, and cylinders.Particularly when the substrate is used in the form of a sheet, such aswhen placed on the surface of a glass microscope slide, defined portionsof the sheet may be apportioned for different molecules of acombinatorial library, as disclosed in Fodor et al., supra. Preferably,the substrate as used in the present invention is formed into particlesthat occupy no more than about 0.0009 mm³, such as a sphere having adiameter of 120 microns, each of which has associated thereto a singlemolecule structure. More preferred, the substrate used in the presentinvention is a bead or sphere having a diameter that is from about 10microns to about 120 microns. Most preferred, the substrate used in thepresent invention is a bead or sphere having a diameter that is fromabout 20 microns to about 80 microns.

[0056] The following examples further illustrate the present inventionand, of course, should not be construed in any way as limiting itsscope.

EXAMPLE 1

[0057] This example illustrates one embodiment of the present inventionwherein a molecular solid covalently linked to a substrate is desorbedfrom the substrate by bombardment of energized particles resulting in afree uncharged molecule, the resultant free uncharged molecule isionized by a laser beam, thus forming a secondary ionized particle, andthe molecular weight of such a secondary ionized particle is determinedby mass spectroscopy.

[0058] Self-assembled monolayers (SAMs) were prepared by immersing vapordeposited gold substrates in a 30 millimolar solution ofphenethylmercaptan (PEM) [C₆H₅CH₂CH₂SH] in ethanol in accordance withNuzzo et al., J. Am. Chem. Soc., 109, 733 (1987). The gold substrateswere kept in solution or at least five days prior to use and rinsed withethanol before introduction into the ultra high voltage (UHV) analysischamber. Thiols adsorb to gold by a strong S-Au bond and are stable inair and vacuum.

[0059] The SAMs were analyzed using a mass spectrometric system asdisclosed in U.S. Pat. No. 5,272,338. Molecules desorbed by 8 keV 1 μspulse of Ar⁺ or H₂ ⁺ were ionized using a 6 ns pulsed laser beam of 266nm photons (3 mJ/pulse) located approximately 1 cm above the surface.The density of molecules in the laser plane was recorded as a functionof time by varying the delay between the primary ion beam impact and thelaser pulse. Analysis of the ionized particles was achieved bytime-of-flight mass spectrometry using a gated detector to select theion of interest. The distribution of flight times from the surface tothe laser was recorded while monitoring m/z 105 [C₆H₅CH₂CH₂ ⁺], whichwas the most abundant ion in the mass spectrum. No molecular ion wasobserved during sputtering or during a gas phase multiphoton ionization(MPI) in which PEM vapor was introduced into the chamber. A detaileddescription of the apparatus used is provided in Kobrin et al., Rev.Sci. Inst., 57, 1354 (1986).

[0060] Time-of-flight distributions for phenethylmercatpan desorbed uponbombardment with Ar⁺ and H₂ ⁺ are displayed in FIG. 2, in which they-axis is labeled Intensity and the x-axis is labeled Time-of-Flight(μs). The shape of the distributions obtained using both Ar⁺ and H₂ ⁺projectiles are nearly identical in the region between 20 and 200 μs andthe most probable time to traverse the distance from the surface to thephoton field is 35 μs. The corresponding kinetic energy distribution(flux) for the H₂ ⁺ projectile is represented by the solid points inFIG. 3, in which the y-axis is labeled Intensity and the x-axis islabeled Kinetic Energy (eV). This curve indicates that the desorbedmolecules have thermal translational energies (ca. 0.025 eV) and havebeen fit by a Maxwell-Boltzmann distribution at room temperature usingstandard methods. Cooling the sample to 165 K causes a marked shift tolower energy, which is again described by a Maxwell-Boltzmanndistribution. The dependence of the desorbed molecule kinetic energy onsubstrate temperature was observed for both projectiles. Thetime-of-flight axis was transformed to kinetic energy under theassumption that [C₆H₅CH₂CH₂] (105 amu) was the molecule desorbed fromthe surface. It is possible that the entire PEM molecule was desorbedand that photofragmentation to form m/z 105 occurred during theionization process. Such a scenario would cause a shift toward aslightly higher kinetic energies, however, the trends observed as afunction of surface temperature would still hold.

[0061] A peak centered at approximately 7 μs is evident in the PEMtime-of-flight distribution produced by the Ar⁺ projectile (FIG. 2).This peak has nearly the same position in time as that of the sputteredgold dimer (FIG. 4, labeled Intensity on the y-axis and Time-of-Flight(μs) on the x-axis), indicating that the molecules in the high energycomponent of the PEM distribution have velocities nearly identical tothat of sputtered Au₂. No gold signal was observed while using H₂ ⁺ as aprojectile.

[0062] Almost all of the desorbed molecules leave the surface withthermal kinetic energies. This result is surprising in view of the factthat the PEM molecules are bound to the surface through a S-Au bondestimated to have an energy of 2 eV (Kobrin et al. supra). Thisobservation is consistent with the presumption that the energy impartedto the gold substrate has no effect on the low energy, high intensityportion of the time-of-flight distribution, which is indicated becausethe distribution profile is independent of projectile mass.

[0063] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A method of identifying a molecule of amolecule-substrate complex, wherein the molecule is covalently attacheddirectly to a substrate or indirectly by means of a linking moiety,comprising: (a) bombarding the molecule-substrate complex with energizedparticles to cleave the molecule from the molecule-substrate complex;and (b) determining the molecular weight of the cleaved molecule bymeans of mass spectrometry.
 2. The method of claim 1 , furthercomprising irradiating the cleaved molecule with photons.
 3. The methodof claim 1 , wherein the molecule is selected from the group consistingof amino acids, peptides, oligonucleotides, heterocyclic compounds, andcombinations thereof.
 4. The method of claim 1 , wherein the substratecomprises a polymeric resin or a metal.
 5. The method of claim 4 ,wherein the polymeric resin is a polystyrene resin having a reactivegroup attached thereto.
 6. The method of claim 1 , wherein the linkingmoiety comprises at least one reactive group that is selected from thegroup consisting of hydroxyl, amino, carboxyl, acetal, thioacetal,C₁-C₁₀ alkylamino, C₁-C₁₀ aralkylamino, and C₁-C₁₀ haloalkyl, and ano-nitrobenzylic group having a benzylic hydrogen.
 7. The method of claim6 , wherein the linking moiety is selected from the group consisting ofF-moc-2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine,F-moc-methoxy-4′(gamma-carboxypropylcxy)benzhydrylamine, p-alkoxybenzylalcohol, benzylacetal, benzylthioacetal, benzhydrylamine, Cl—CH₂—Ph,2-methoxy-4-alkoxy benzyl alcohol, and o-nitrobenzyloxy carbonyl.
 8. Themethod of claim 7 , wherein the linking moiety is selected from thegroup consisting of 2-methoxy-4-alkoxy benzyl alcohol, benzylacetal, andbenzylthioacetal.
 9. The method of claim 8 , wherein the covalent bondis cleaved without substantial modification of the molecule.
 10. Themethod of claim 9 , wherein the substrate is a bead.
 11. The method ofclaim 10 , wherein the bead has a diameter of from about 10 microns toabout 120 microns.
 12. The method of claim 10 , wherein the massspectrometry is time-of-flight secondary ion mass spectrometry.
 13. Themethod of claim 12 , wherein the method further comprises mapping of thespatial distribution of the molecule amidst a plurality ofmolecule-substrate complexes.
 14. The method of claim 11 , wherein themolecule is an amino acid or a peptide.
 15. The method of claim 14 ,wherein the peptide comprises two to ten amino acids.
 16. The method ofclaim 15 , wherein the method further comprises determination of thesequence of the peptide from the fragmentation pattern obtained in themass spectrometry.
 17. The method of claim 12 , wherein the molecule isa heterocyclic compound comprising four to seven membered rings havingN, S, or O, and combinations thereof.
 18. The method of claim 1 ,wherein the particles are energized by subjection to an electric fieldof between about one to about 30 kilovolts.
 19. The method of claim 18 ,wherein the particles are gallium or argon.
 20. The method of claim 1 ,wherein the irradiated molecule becomes ionized.
 21. The method of claim2 , wherein the substrate is a polystyrene bead having a reactive group,the molecule is an amino acid, peptide, oligonucleotide, or aheterocyclic compound, or a combination thereof, the energized particlesare gallium atoms, the photon source is a laser, and the massspectrometry is time-of-flight secondary ion mass spectrometry.